The development of novel herbicide resistance in plants offers significant production and economic advantages. As one example, rice production is frequently restricted by the prevalence of a weedy relative of rice that flourishes in commercial rice fields. The weed is commonly called “red rice,” and belongs to the same species as cultivated rice (Oryza sativa L.). The genetic similarity of red rice and commercial rice has made herbicidal control of red rice difficult. The herbicides Ordram™ (molinate: S-ethyl hexahydro-1-H-azepine-1-carbothioate) and Bolero™ (thiobencarb: S-[(4-chlorophenyl)methyl]diethylcarbamothioate) offer partial suppression of red rice, but no herbicide that actually controls red rice is currently used in commercial rice fields because of the simultaneous sensitivity of existing commercial cultivars of rice to such herbicides. The release of mutant commercial rice lines having resistance to herbicides that are effective on red rice will greatly increase growers' ability to control red rice infestations. The development of herbicide resistance in other crops and other plants will have similar benefits.
Rice producers in the southern United States typically rotate rice crops with soybeans to help control red rice infestations. While this rotation is not usually desirable economically, it is frequently necessary because no herbicide is currently available to control red rice infestations selectively in commercial rice crops. During the soybean rotation, the producer has a broad range of available herbicides that may be used on red rice, so that rice may again be grown the following year. United States rice producers can lose $200-$300 per acre per year growing soybeans instead of rice, a potential loss affecting about 2.5 million acres annually. Additional losses in the United States estimated at $50 million per year result from the lower price paid by mills for grain shipments contaminated with red rice. Total economic losses due to red rice in the southern United States alone are estimated to be $500 to $750 million a year. Economic losses due to red rice may be even greater in other rice-producing countries.
Rice producers typically use the herbicides propanil (trade name Stam™) or molinate (trade name Ordram™) to control weeds in rice production. Propanil has no residual activity. Molinate is toxic to fish. Neither of these herbicides controls red rice. Imazethapyr ((±)-2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl-3-pyridinecarboxylic acid) offers an environmentally acceptable alternative to molinate, has the residual weed control activity that propanil lacks, and is a very effective herbicide on red rice. Imazethapyr also offers excellent control of other weeds important in rice production, including barnyardgrass. Barnyardgrass is a major weed in rice production, and is currently controlled with propanil or molinate. However, barnyardgrass has developed resistance to propanil in some regions.
The total potential market for rice varieties that are resistant to a herbicide that can control red rice is about 5.3 million acres in the United States, and the potential market outside the United States is much larger. World rice production occupies about 400 million acres. Red rice and other weeds are major pests in rice production in the United States, Brazil, Australia, Spain, Italy, North Korea, South Korea, Philippines, Vietnam, China, Taiwan, Brazil, Argentina, Colombia, India, Pakistan, Bangladesh, Japan, Ecuador, Mexico, Cuba, Malaysia, Thailand, Indonesia, Sri Lanka, Venezuela, Myanmar, Nigeria, Uruguay, Peru, Panama, Dominican Republic, Guatemala, Nicaragua, and other rice-producing areas.
A number of herbicides target acetohydroxyacid synthase (AHAS), an enzyme also known as acetolactate synthase (ALS), and also designated as E. C. 4.1.3.18. This enzyme catalyzes the first step in the synthesis of the amino acids leucine, valine, and isoleucine. Inhibition of the AHAS enzyme is normally fatal to plants. Herbicides that inhibit the enzyme acetohydroxyacid synthase would offer a number of advantages over currently available herbicides if they could be used in commercial rice production, and the production of other crops, in circumstances where they could not otherwise be used. Potential advantages include long residual activity against weeds, effective control of the more important weeds in rice production, including red rice, and relative environmental acceptability. Even in regions where red rice is not currently a problem, the availability of herbicide-resistant rice can have a major influence on rice production practices by providing the farmer with a new arsenal of herbicides suitable for use in rice fields.
Total potential demand for resistance to AHAS-acting herbicides in plants other than rice is difficult to estimate, but could be very large indeed.
U.S. Pat. No. 4,761,373 describes the development of mutant herbicide-resistant maize plants through exposing tissue cultures to herbicide. The mutant maize plants were said to have an altered enzyme, namely acetohydroxyacid synthase, that conferred resistance to certain imidazolinone and sulfonamide herbicides. See also U.S. Pat. Nos. 5,304,732, 5,331,107, 5,718,079, 6,211,438, 6,211,439, and 6,222,100; and European Patent Application 0 154 204 A2.
Lee et al., “The Molecular Basis of Sulfonylurea Herbicide Resistance in Tobacco,” The EMBO J., vol. 7, no. 5, pp. 1241-1248 (1988), describe the isolation and characterization from Nicotiana tabacum of mutant genes specifying herbicide resistant forms of acetolactate synthase, and the reintroduction of those genes into sensitive lines of tobacco.
Saxena et al., “Herbicide Resistance in Datura innoxia,” Plant Physiol., vol. 86, pp. 863-867 (1988) describe several Datura innoxia lines resistant to sulfonylurea herbicides, some of which were also found to be cross-resistant to imidazolinone herbicides.
Mazur et al., “Isolation and Characterization of Plant Genes Coding for Acetolactate Synthase, the Target Enzyme for Two Classes of Herbicides,” Plant Physiol. vol. 85, pp. 1110-1117 (1987), discuss investigations into the degree of homology among acetolactate synthases from different species.
U.S. Pat. No. 5,767,366 discloses transformed plants with genetically engineered imidazolinone resistance, conferred through a gene cloned from a plant such as a mutated Arabidopsis thaliana. See also a related paper, Sathasivan et al., “Nucleotide Sequence of a Mutant Acetolactate Synthase Gene from an Imidazolinone-resistant Arabidopsis thaliana var. Columbia,” Nucleic Acids Research vol. 18, no. 8, p. 2188 (1990).
Examples of resistance to AHAS-inhibiting herbicides in plants other than rice are disclosed in U.S. Pat. No. 5,013,659; K. Newhouse et al., “Mutations in corn (Zea mays L.) Conferring Resistance to Imidazolinone Herbicides,” Theor. Appl. Genet., vol. 83, pp. 65-70 (1991); K. Sathasivan et al., “Molecular Basis of Imidazolinone Herbicide Resistance in Arabidopsis thaliana var Columbia,” Plant Physiol. vol. 97, pp. 1044-1050 (1991); B. Miki et al., “Transformation of Brassica napus canola cultivars with Arabidopsis thaliana Acetohydroxyacid Synthase Genes and Analysis of Herbicide Resistance,” Theor. Appl. Genet., vol. 80, pp. 449458 (1990); P. Wiersma et al., “Isolation, Expression and Phylogenetic Inheritance of an Acetolactate Synthase Gene from Brassica napus,” Mol. Gen. Genet., vol. 219, pp. 413-420 (1989); J. Odell et al., “Comparison of Increased Expression of Wild-Type and Herbicide-Resistant Acetolactate Synthase Genes in Transgenic Plants, and Indication of Postranscriptional Limitation on Enzyme Activity,” Plant Physiol., vol. 94, pp. 1647-1654 (1990); published international patent application WO 92/08794; U.S. Pat. No. 5,859,348; published international patent application WO 98/02527; published European Patent Application EP 0 965 265 A2, and published international patent application WO 90/14000.
U.S. Pat. Nos. 5,853,973 and 5,928,937 disclose the structure-based modeling of AHAS, the presumptive binding pockets on the enzyme for AHAS-acting herbicides, and certain designed AHAS mutations to confer herbicide residence. These patents also disclose amino acid sequences for the AHAS enzymes and isozymes from several plants, including that from Zea mays. See also published international patent application WO 96/33270.
S. Sebastian et al., “Soybean Mutants with Increased Tolerance for Sulfonylurea Herbicides,” Crop. Sci., vol. 27, pp. 948-952 (1987) discloses soybean mutants resistant to sulfonylurea herbicides. See also U.S. Pat. No. 5,084,082.
K. Shimamoto et al., “Fertile Transgenic Rice Plants Regenerated from Transformed Protoplasts,” Nature, vol. 338, pp. 274-276 (1989) discloses a genetic transformation protocol in which electroporation of protoplasts was used to transform a gene encoding β-glucuronidase into rice.
T. Terakawa et al., “Rice Mutant Resistant to the Herbicide Bensulfuron Methyl (BSM) by in vitro Selection,” Japan. J. Breed., vol. 42, pp. 267-275 (1992) discloses a rice mutant resistant to a sulfonylurea herbicide, derived by selective pressure on callus tissue culture. Resistance was attributed to a mutant AHAS enzyme.
Following are publications by the inventor (or the inventor and other authors) concerning research on herbicide-resistant rice varieties. These publications are T. Croughan et al., “Rice and Wheat Improvement through Biotechnology,” 84th Annual Research Report, Rice Research Station, 1992, pp. 100-103 (1993); T. Croughan et al., “Rice and Wheat Improvement through Biotechnology,” 85th Annual Research Report, Rice Research Station, 1993, pp. 116-156 (1994); T. Croughan, “Application of Tissue Culture Techniques to the Development of Herbicide Resistant Rice,” Louisiana Agriculture, vol. 37, no. 3, pp. 25-26 (1994); T. Croughan et al., “Rice Improvement through Biotechnology,” 86th Annual Research Report, Rice Research Station, 1994, pp. 461-482 (1995); T. Croughan et al., “Assessment of Imidazolinone-Resistant Rice,” 87th Annual Research Report, Rice Research Station, 1994, pp. 491-525 (September 1996); T. Croughan et al., “IMI-Rice Evaluations,” 88th Annual Research Report, Rice Research Station, 1996, pp. 603-629 (September, 1997); T. Croughan et al., “Rice Biotechnology Research,” 89th Annual Research Report, Rice Research Station, 1997, p. 464 (September, 1998); T. Croughan et al., “Imidazolinone-Resistant Rice,” 90th Annual Research Report, Rice Research Station, 1998, p. 511 (December, 1999); T. Croughan et al., “Rice and Wheat Improvement through Biotechnology,” USDA CRIS Report Accession No. 0150120 (for Fiscal Year 1994—actual publication date currently unknown); T. Croughan, “Improvement of Lysine Content and Herbicide Resistance in Rice through Biotechnology,” USDA CRIS Report Accession No. 0168634 (for Fiscal Year 1997—actual publication date currently unknown); T. Croughan, “Improvement of Lysine Content and Herbicide Resistance in Rice through Biotechnology,” USDA CRIS Report Accession No. 0168634 (for Fiscal Year 1999—actual publication date currently unknown); T. Croughan, “Improvement of Lysine Content and Herbicide Resistance in Rice through Biotechnology,” USDA CRIS Report Accession No. 0168634 (for Fiscal Year 2000—actual publication date currently unknown); T. Croughan, “Herbicide Resistant Rice,” Proc. 25th Rice Tech. Work. Groups, p. 44 (1994); T. Croughan et al., “Applications of Biotechnology to Rice Improvement,” Proc. 25th Rice Tech. Work. Groups, pp. 62-63 (1994); T. Croughan, “Production of Rice Resistant to AHAS-Inhibiting Herbicides,” Congress on Cell and Tissue Culture, Tissue Culture Association, In Vitro, vol. 30A, p. 60, Abstract P-1009 (Jun. 4-7, 1994). (Note that the Annual Research Reports of the Rice Research Station are published in the year after the calendar year for which activities are reported. For example, the 84th Annual Research Report, Rice Research Station, 1992, summarizing research conducted in 1992, was published in 1993.) The reports in the 87th and 88th Annual Research Report, Rice Research Station (published September, 1996 and September, 1997, respectively) mention the breeding line 93AS3510 in tables giving data on certain herbicide resistance trials. These reports gave no information on how the breeding line was developed. The breeding line was not publicly available at the times these reports were published. The breeding line 93AS3510 is the same as the ATCC 97523 rice that is described in greater detail in the present inventor's later-published international patent application WO 97/41218 (1997) and U.S. Pat. Nos. 5,736,629, 5,773,704, 5,952,553, and 6,274,796.
See also E. Webster et al., “Weed Control Systems for 1 ml-Rice,” p. 33 in Program of the 27th Rice Technical Working Group Meeting (March, 1998); L. Hipple et al., “AHAS Characterization of Imidazolinone Resistant Rice,” pp. 4546 in Program of the 27th Rice Technical Working Group Meeting (March, 1998); W. Rice et al., “Delayed Flood for Rice Water Weevil Control using Herbicide Resistant Germplasm,” p. 61 in Program of the 27th Rice Technical Working Group Meeting (March 1998); E. Webster et al., “Weed Control Systems for Imidazolinone-Rice,” p. 215 in Proceedings of the 27th Rice Technical Working Group Meeting (1999); L. Hipple et al., “AHAS Characterization of Imidazolinone Resistant Rice,” pp. 68-69 in Proceedings of the 27th Rice Technical Working Group Meeting (1999); W. Rice et al., “Delayed Flood for Rice Water Weevil Control using Herbicide Resistant Germplasm,” p. 134 in Proceedings of the 27th Rice Technical Working Group Meeting (1999); and W. Rice et al., “Delayed flood for management of rice water weevil (Coleopterae: Curculionidae),” Environmental Entomology, vol. 28, no. 6, pp. 1130-1135 (December, 1999).
The present inventor's U.S. Pat. No. 5,545,822 discloses a line of rice plants having a metabolically-based resistance to herbicides that interfere with the plant enzyme acetohydroxyacid synthase; i.e., the herbicide resistance of these rice plants was not due to a resistant AHAS enzyme. (See published international patent application WO 97/41218, pages 6-9.) See also the present inventor's U.S. Pat. No. 5,773,703.
The present inventor's published international patent application WO 97/41218 discloses one line of rice plants having a mutant AHAS enzyme that is resistant to herbicides that interfere with the wild-type plant enzyme acetohydroxyacid synthase. This line of rice plants was developed by exposing rice seeds to the mutagen methanesulfonic acid ethyl ester (EMS), and screening millions of progeny for herbicide resistance. See also the present inventor's U.S. Pat. Nos. 5,736,629, 5,773,704, 5,952,553, and 6,274,796.
The present inventor's published international patent application WO 00/27182 discloses additional lines of rice plants having mutant AHAS enzymes that are resistant to herbicides that interfere with the wild-type plant enzyme acetohydroxyacid synthase. These lines of rice plants were developed by exposing rice seeds to EMS, and screening millions of progeny for herbicide resistance.
U.S. Pat. No. 4,443,971 discloses a method for preparing herbicide tolerant plants by tissue culture in the presence of herbicide. U.S. Pat. No. 4,774,381 discloses sulfonylurea (sulfonamide) herbicide-resistant tobacco plants prepared in such a manner.
U.S. Pat. No. 5,773,702 discloses sugar beets with a resistant mutant AHAS enzyme, derived from cell cultures grown in the presence of herbicide. See also published international patent application WO 98/02526.
Published international patent application WO 00/26390 discloses the cloning and sequencing of the Arabidopsis AHAS small subunit protein, and an expression vector to transform plants with that small AHAS subunit to impart herbicide tolerance.
U.S. Pat. No. 5,633,437 discloses a herbicide resistant AHAS enzyme and gene isolated from cockleburs.
U.S. Pat. No. 5,767,361 discloses a mutant, resistant AHAS enzyme from maize. The definitions of the 5,767,361 patent are incorporated into the present disclosure by reference, to the extent that those definitions are not inconsistent with the present disclosure, as are that patent's descriptions of certain genetic transformation techniques for plants. See also U.S. Pat. No. 5,731,180 and European Patent Application 0 525 384 A2.
U.S. Pat. No. 5,605,011; European Patent Application 0 257 993 A2; and European Patent Application 0 730 030 A1 disclose resistant acetolactate synthase enzymes derived from callus culture of tobacco cells in the presence of herbicide, from spontaneous mutations of the ALS gene in yeast; EMS-induced mutations in Arabidopsis seeds; certain modifications of those enzymes; and the transformation of various plants with genes encoding the resistant enzymes. These patents disclose several techniques for modifying AHAS genes to produce herbicide-resistant AHAS enzymes, and for transforming plants with those genes.
U.S. Pat. No. Re 35,661 (a reissue of U.S. Pat. No. 5,198,599) discloses lettuce plants with enhanced resistance to herbicides that target the enzyme acetolactate synthase. The initial source of herbicide resistance was a prickly lettuce weed infestation in a grower's field, an infestation that was not controlled with commercial sulfonylurea herbicides.
T. Shimizu et al., “Oryza sativa ALS mRNA for acetolactate synthase, complete cds, herbicide sensitive wild type,” BLAST accession number AB049822 (April 2001), available through www.ncbi.nlm.nih.gov/blast, discloses the nucleotide sequence and inferred amino acid sequence for wild type ALS cDNA from Oryza sativa var. Kinmaze. These two sequences are reproduced below as SEQ ID NOS 2 and 3, respectively.
T. Shimizu et al., “Oryza sativa ALS mRNA for acetolactate synthase, complete cds, herbicide resistant biotype,” BLAST accession number AB049823 (April 2001), available through www.ncbi.nlm.nih.gov/blast, discloses the nucleotide sequence and inferred amino acid sequence for an ALS cDNA from Oryza sativa var. Kinmaze that was reported to be herbicide resistant, although the nature of the herbicide resistance is not specified in the BLAST description. These two sequences are reproduced below as SEQ ID NOS 4 and 5, respectively.
Following are selected data taken from some of the references cited above, concerning the locations of certain imidazolinone or sulfonylurea herbicide tolerance mutations in AHAS/ALS from various species. No attempt has been made to reconcile or align the different nucleotide or amino acid numbering systems used in the different references. In describing the substitutions below (as well as in the remainder of the specification and the claims), the wild-type nucleotide or amino acid is always listed first, followed by the mutant nucleotide or amino acid. All substitutions discussed refer to the AHAS/ALS DNA coding sequence, or to the expressed or inferred AHAS/ALS amino acid sequence.
Lee et al. (1988) reported that there were two homologous ALS genes in Nicotiana tabacum. The sulfonylurea herbicide-resistant C3 mutant in one ALS gene had a Pro-Gln replacement at amino acid 196; while the sulfonylurea herbicide-resistant S4-Hra mutant in the other ALS gene had two amino acid changes: Pro-Ala at amino acid 196, and Trp-Leu at amino acid 573.
Sathasivan et al. (1990), Sathasivan et al. (1991), and U.S. Pat. No. 5,767,366 reported a G-A nucleotide substitution at position 1958, corresponding to a Ser-Asn substitution at position 653, in an imidazolinone herbicide-resistant Arabidopsis thaliana. 
Wiersma et al. (1989) reported sulfonylurea herbicide resistance in tobacco plants that had been transformed with a mutant Brassica napus ALS gene, in which codon 173 had been altered by site-directed mutagenesis to replace Pro with Ser.
European patent application 0 257 993 A2 reported several spontaneous mutations in the yeast (Saccharomyces cerevisiae) ALS gene that resulted in sulfonylurea herbicide resistance: at amino acid position 121, a substitution of wild-type Gly by Ser; at amino acid position 122, a substitution of wild-type Ala by Pro, Asp, Val, or Thr; at position 197, a substitution of wild-type Pro by Ser or Arg; at position 205, a substitution of wild-type Ala by Asp or Thr; at position 256, a substitution of wild-type Lys by Glu, Thr, or Asn; at position 359, a substitution of wild-type Met by Val; at position 384, a substitution of wild-type Asp by Glu, Val, or Asn; at position 588, a substitution of wild-type Val by Ala; at position 591, a substitution of wild-type Trp by Arg, Cys, Gly, Leu, Ser, or Ala; at position 595, a substitution of wild-type Phe by Leu. The same patent application reported several site-directed mutations of the yeast ALS gene at some of the same positions to also produce sulfonylurea herbicide resistance: at amino acid position 122, a substitution of wild-type Ala by Ser, Val, Thr, Pro, Asn, Ile, His, Arg, Leu, Tyr, Cys, Phe, Glu, Met, Lys, Gln, or Trp; at position 205, a substitution of wild-type Ala by Arg, Cys, Glu, or Trp; at position 256, a substitution of wild-type Lys by Asp, Gly, Leu, Pro, or Trp; at position 359, a substitution of wild-type Met by Pro or Glu; at position 384, a substitution of wild-type Asp by Pro, Trp, Ser, Gly, Cys, or Lys; at position 591, a substitution of wild-type Trp by Asp, Glu, Phe, His, Tyr, Ile, Val, Lys, Arg, Met, Asn, Gln, or Thr. See also U.S. Pat. No. 5,605,011, which also describes experimental data for the following site-directed mutations: at amino acid 121, a substitution of wild-type Gly by Asn or Ala; at amino acid 197, a substitution of wild-type Pro by Gln, Glu, Ala, Gly, Trp, Tyr, Cys, or Val; at amino acid 205, a substitution of wild-type Ala by Tyr, Val, or Asn; at amino acid 359, a substitution of wild-type Met by Gln, Lys, Tyr, or Cys; at position 583, a substitution of wild-type Val by Ser, Asn, Trp, or Cys; and at position 595, a substitution of wild-type Phe by Gly, Asn, Arg, Cys, Pro, Ser, or Trp. Other amino substitutions at the same positions are also described prophetically, without experimental data. See also U.S. Pat. No. 5,013,659.
WO 98/02527 reported sulfonylurea and triazolopyrimidine resistance in one line of sugar beets resulting from a C-T substitution at nucleotide 562, corresponding to a Pro-Ser substitution at amino acid 188. This same reference also reported sulfonylurea, imidazolinone, and triazolopyrimidine resistance in a second line of sugar beets resulting from two mutations: The same mutation as reported in the first line (from which the second line had been derived), coupled with a G-A substitution at nucleotide 337, corresponding to an Ala-Thr substitution at amino acid 113. See also WO 98/02526, U.S. Pat. Nos. 5,859,348 and 5,773,702.
WO 96/33270 describes a number of designed or predicted mutations from a structure-based modeling method, that were said to induce imidazolinone tolerance in AHAS Experimental results confirming such tolerance in mutated Arabidopsis AHAS, either in vitro or in transformed tobacco plants in vivo, were provided for the following substitutions: Met-Ile at amino acid position 124, Met-His at position 124, Arg-Glu at position 199, and Arg-Ala at position 199. See also U.S. Pat. Nos. 5,928,937 and 5,853,973.
WO 92/08794 reported imidazolinone resistance in two lines of maize. One had a G-A substitution at nucleotide position 171, resulting in an Ala-Thr substitution at the corresponding amino acid position. The other had a G-A substitution at position 1888, resulting in a Ser-Asn substitution at the corresponding amino acid position.
U.S. Pat. No. 5,731,180 reported imidazolinone resistance in maize resulting from a G-A substitution at nucleotide position 1898, resulting in a Ser-Asn substitution at amino acid position 621. See also U.S. Pat. No. 5,767,361 and European patent application 0 525 384.
U.S. Pat. No. 5,633,437 reported imidazolinone resistance in cockleburs, characterized by five differences between resistant ALS enzyme biotypes and sensitive biotypes: Lys-Glu at amino acid position 63, Phe-Leu at position 258, Gln-His at position 269, Asn-Ser at position 522, and Trp-Leu at position 552. The changes at positions 522 and 552 were thought to be particularly important.
T. Shimizu et al., “Oryza sativa ALS mRNA for acetolactate synthase, complete cds, herbicide resistant biotype,” BLAST accession number AB049823 (April 2001) reported a nucleotide sequence and inferred amino acid sequence for a rice ALS that was said to be herbicide resistant, although the nature of the herbicide resistance was not specified in the BLAST entry. As compared to a contemporaneous wild type ALS for the same rice variety (Kinmaze), the inferred amino acid sequence for the resistant ALS appeared to display two differences: a Trp-Leu substitution at position 548, and a Ser-Ile substitution at position 627.